Influenza A viruses are divided into subtypes on the basis of the antigenicity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); influenza viruses bearing 15 HA and 9 NA subtypes have been isolated from birds, but only H1N1, H2N2, and H3N2 subtype viruses have circulated widely and caused epidemic disease in humans in the last century. Aquatic birds serve as a reservoir from which new subtypes of influenza A viruses enter the human population. In the last 10 years, human infections with avian influenza viruses (AIV) of three subtypes, H7, H5 and H9, have been detected on several occasions, accompanied in each case by contemporaneous outbreaks of disease in poultry. In 2003, a large outbreak of human infections with a highly pathogenic avian influenza H7N7 virus occurred in Europe. On several occasions since 1997, there have been serious outbreaks caused by a highly pathogenic avian influenza H5N1 viruses in Asia. The most recent H5N1 outbreak in poultry began in late 2003 and affected at least 10 countries in Asia. Recent reports indicate that H5N1 viruses have been isolated from migratory and wild birds in China, Russia, Kazakhstan, Mongolia and Tibet. Human cases of H5N1 infection have been reported since December 2003 in Indonesia, Vietnam, Thailand and Cambodia with a total of 112 cases and 57 deaths as of August 5, 2005. There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including formalin inactivated whole or split virus, HA subunit, and live attenuated vaccines. Live attenuated vaccines generally induce broadly cross-reactive protection, which may be a useful feature in the event of a pandemic if a vaccine generated from the actual pandemic strain is not available. The goal of our program is to generate candidate live attenuated reassortant influenza virus vaccines against a range of influenza A subtypes that have pandemic potential and to evaluate these vaccines in preclinical studies and clinical trials. The vaccine viruses will contain the hemagglutinin (HA) and neuraminidase (NA) genes of a selected avian influenza virus with pandemic potential and the attenuating genes from the A/Ann Arbor/6/60 cold adapted (A/AA/6/60 ca) donor virus. The cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (AA) (H2N2) has been developed as a live attenuated vaccine seed virus that exhibits cold-adaptation, temperature-sensitive (ts), and attenuation (att) phenotypes which are specified by mutations in the internal genes. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of the AA ca virus have been repeatedly demonstrated to bear these phenotypes and extensive evaluation in humans has proven them to be attenuated and safe as live virus vaccines. This approach has been licensed for general use for interpandemic influenza A and B virus infections. It is not known whether the candidate vaccine viruses bearing avian influenza HA and NA genes will be over-attenuated for humans or will be associated with some residual virulence. Live attenuated vaccines must be able to replicate to levels that elicit a protective immune response without causing disease in the host so a balance between attenuation and infectivity must be achieved. An optimal public health response in the event of a potential pandemic requires that vaccines be available to prevent infection with minimum delay and an important approach to pandemic preparedness is to generate and evaluate candidate vaccines against influenza A subtypes that are recognized to have pandemic potential, prior to their actual spread. We generated a candidate H9N2 influenza vaccine by genetic reassortment; the vaccine strain contains the hemagglutinin and neuraminidase genes from an avian H9N2 influenza virus and six internal gene segments from the AA ca virus. The candidate H9N2 vaccine virus demonstrated the ts, ca and att phenotypes of the AA ca vaccine donor virus. The H9N2 AA ca vaccine virus was immunogenic in mice and protected mice from subsequent challenge with homologous and heterologous H9N2 viruses. A clinical lot of this vaccine was generated and a Phase I clinical trial of the safety and immunogenicity of the vaccine for healthy adults was undertaken under an IND. Analysis of laboratory results from the clinical trial is under way. In order to generate candidate vaccines against H5N1 viruses that have caused human infections in Asia in 1997, 2003 and 2004, we applied plasmid based reverse genetics, a technique in which infectious virus can be recovered from cells co-transfected with plasmids expressing each of the 8 influenza gene segments, to generate reassortant viruses that contain the hemagglutinin and neuraminidase genes from H5N1 influenza viruses and six internal gene segments from the AA ca virus. We removed the virulence motif of multiple basic amino acid motifs in the hemagglutinin gene of the highly pathogenic H5N1 influenza virus that are associated with pathogenicity in poultry. The ts and ca phenotypes of the candidate pandemic vaccines will be evaluated in vitro and preclinical studies will be carried out in mice to determine the level of attenuation, immunogenicity and efficacy against challenge. Clinical trials of the H5N1 vaccines will focus on safety, infectivity and immunogenicity of the candidate influenza vaccines.